Konomura Naoto, Arai Naoto, Shinohara Takeshi, Kobayashi Jun, Iwasaki Wakana, Ikawa Shukuko, Kusano Kohji, Shibata Takehiko
Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan.
Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.
Nucleic Acids Res. 2017 Jan 9;45(1):337-352. doi: 10.1093/nar/gkw998. Epub 2016 Oct 27.
RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.
RecA家族重组酶催化的ATP依赖性同源接头形成对于同源重组至关重要，其中RecA或Rad51首先与单链（ss）DNA结合，然后与双链（ds）DNA相互作用。然而，当RecA或Rad51在结合ssDNA之前与dsDNA相互作用时，RecA或Rad51的同源接头形成活性会迅速受到抑制。我们发现，在这些以及产生二磷酸腺苷（ADP）的重组酶活性抑制条件下，相似最佳浓度的RecA或Rad51可将DNA连接酶催化的双链DNA末端连接（DNA连接）增强约30至40倍。RecA或Rad51对DNA连接的增强作用可在2至5分钟内将大部分底物DNA转化为多聚体，并且对于这种增强作用，ADP是常见且最佳辅因子。三磷酸腺苷（ATP）对RecA有效，但对Rad51无效。如一个突变体所示，Rad51/RecA增强的DNA连接依赖于双链DNA结合，并且与与DNA连接酶的物理相互作用无关。这些观察结果证明了RecA和Rad51在并列双链DNA末端以准备由DNA连接酶进行共价连接方面的共同和独特活性。Rad51的这种新的体外功能为我们的遗传学观察提供了一个简单解释，即Rad51在体内通过酵母经典非同源末端连接（NHEJ）在报告质粒DNA末端连接的保真度中发挥作用。
